The present disclosure relates to a dielectric cytometric apparatus for analyzing and sorting cells as well as a dielectric cytometric cell sorting method.
In the fields of life sciences and medical researches or in the fields of medical cares such as clinical practices, an analysis method referred to as flow cytometry is adopted. In the flow cytometry, liquid composed of cells individually freed from each other is taken as a sample. Under a dilute condition with an inter-cell average distance sufficiently greater than the dimensions of the cell, liquid serving as a sample is driven to flow through the inside of a flow channel pipe. A signal detection section installed in the flow channel pipe carries out a certain analysis/measurement on individual cells flowing through the signal detection section. Cells having measured signals approximating each other are considered to be cells of the same type. Thus, signals measured for a number of cells included in the sample liquid are analyzed in order to identify the type of cells included in a cell group serving as the sample and calculate a cell-type cell count representing the number of cells included in the cell type. Instead of calculating the number of cells included in the cell type, it is also possible to calculate a ratio of the cell-type cell count to the total number of cells. The analysis method adopted in the flow cytometry is classified into large categories, that is, an optical analysis method and an electrical analysis method.
As the optical analysis method, a combination of only a fluorescent detection method and a light scattering detection method is adopted. The principle of the fluorescent detection method is explained as follows.
On the surface of a cell, there are protein molecules each referred to as a surface antigen. The surface antigen is by no means limited to one type. Thus, by identifying the type of the surface antigen and the number of surface antigens included in the type of the antigen, it is possible to identify the cell type to which the cells pertain. If the surface-antigen molecules are known, it is possible to synthesize molecules, which are specifically joinable to the surface-antigen molecules, to the surface-antigen molecules. The molecules specifically joinable to the surface-antigen molecules are referred to as antibody molecules for the surface antigens. In addition, it is also possible to chemically join fluorescent labeling molecules to the antibody molecules. A fluorescent labeling molecule is a molecule which generates fluorescent light if light having a wavelength in a specific wavelength band is radiated to the molecule. That is to say, fluorescent labeling antibodies generating fluorescent light beams having different wavelengths are each synthesized with a surface antigen molecule used for characterizing the cell type assumed to be included in a cell group serving as a subject of an analysis. The composite of all these fluorescent labeling antibodies is taken as a labeling test reagent. If this labeling test reagent is added to liquid solution, each of cells of the labeling test reagent is labeled with a fluorescent molecule which varies from cell type to cell type to which the cells pertain.
In a signal detection section installed in a flow channel pipe of a flow cytometric apparatus also referred to as a flow cytometer, laser light is radiated to a cell passing through the signal detection section. When laser light is radiated to such cells, the surface-antigen molecules of individual cells and fluorescent labeling molecules joined to antibody molecules specifically joined to the surface-antigen molecules are excited, generating fluorescent light having a wavelength peculiar to the fluorescent labeling molecules. For a number of cells, the fluorescent light is detected in order to count the number of cells for every cell type. This method is adopted widely. As a matter of fact, the so-called flow cytometry is intended to imply essentially this method.
The flow cytometer put into the market is used not only for obtaining the existing state of surface antigen molecules but also additional information such as cell dimensions and the internal density of the cells. Thus, the flow cytometer is used for measuring the strength of laser light scattered by cells at the same time.
An electrical method has been put to practical use as the method of a Coulter counter. For more information on this Coulter counter, the reader is advised to refer to documents such as U.S. Pat. No. 2,656,508. In the Coulter counter, a pair of electrodes are provided on a signal detection section inside a flow channel pipe. A voltage is applied between the electrodes. When an individual cell passes through the space between the electrodes, the resistance of the space changes. A frequency at which the resistance changes is measured in order to count the number of cells passing through the signal detection section. In addition, the magnitude of the change of the resistance is approximately proportional to the volume of the cell. Thus, if a cell group serving as the object of the analysis includes cells of different types having dimensions much different from each other, the operation to count the number of cells can be carried out for each of the cell types.
As an improved technology of the Coulter counter, there has been proposed a technology of superposing an AC (Alternating Current) voltage having a frequency of tens of MHz on a DC (Direct Current) voltage applied between the electrodes. For more information on this improved technology, the reader is advised to refer to documents such as U.S. Pat. Nos. 3,502,974 and 6,204,668. As is commonly known, there is a correlation between the AC resistance of the cell at frequencies of tens of MHz and the internal density of the cell. By obtaining measured data for each of the AC and DC resistances, a detailed analysis can be conducted in comparison with the existing methods.
The electrical analysis method based on only a DC resistance or a combination of a DC resistance and an AC resistance is used in some flow cytometers by combining the electrical analysis method with the optical analysis method. In addition, in the clinical examination field, there is used an automatic blood-cell counting apparatus for counting, among others, the number of red-blood cells, the number of white-blood cells and the number of blood platelets. Normally, the automatic blood-cell counting apparatus is differentiated from the flow cytometer. From the standpoint of the operation principle of the automatic blood-cell counting apparatus, however, the automatic blood-cell counting apparatus can be said to be a flow cytometer in a broader sense. In this specification, without differentiating the automatic blood-cell counting apparatus and the flow cytometer from each other, the technical term “flow cytometer” is used to imply both the automatic blood-cell counting apparatus and the automatic blood-cell counting apparatus which is a flow cytometer in a broader sense.
As described above, in the present state of the art, the flow cytometer adopting the electrical analysis method is implemented by adopting also the optical analysis method.
Next, a cell sorting technology adopted by the flow cytometer is explained.
Applications include not only analyses of cells included in liquid solution, but also possibly sorting of only cells included in a specific cell type from other cells by making use of results of the analyses. For example, there is a case in which a cell type appearing in peripheral blood exists due to a sort of blood cancer. In this case, only the cell type is sorted and a gene analysis or a protein analysis is carried out on the cell type. By conducting such an analysis, it is quite within the bounds of possibility that a clue as to what has caused the blood-cancer disease is obtained. For example, the clue may suggest that the blood-cancer disease has been caused by a gene abnormality or the like. As another example, in an attempt to induce an iPS cell from a cell of a human being, not every cell of the human being is induced into an iPS cell. It is thus necessary to sort only iPS cells from cultured cells.
In these cases, it is necessary to provide a mechanism for sorting only specific cells in accordance with a signal generated by a signal detection section provided in the flow channel pipe on the downstream side of the signal detection section. This mechanism is referred to as a sorter. The sorter is provided in upper-level models of the flow cytometer put into the market.
If a cell sorted by a fluorescent flow cytometer is used for a research purpose, a big problem is raised. In this case, the fluorescent flow cytometer is a flow cytometer based on a fluorescent detection method selected among optical analysis methods. The big problem is that, strictly speaking, the original state of the cell used as the subject of control is different from the state of a fluorescently labeled cell. If an antibody molecule is joined to a surface antigen molecule, chemical excitement is added to the inside of the cell so that a multi-stage signal transmission reaction may probably take place. However, the effect of such a small change can be regarded as a small effect so that the cell can be normally used for a research purpose.
It is to be noted that, as a technology related to the present disclosure, there has been proposed a technology for measuring a dielectric spectrum of every cell and, on the basis of the result of the measurement, cells are sorted. For more information on this technology, the reader is advised to refer to document such as Japanese Patent Laid-open No. 2010-181399. Additionally, the reader is suggested to refer to document such as JP-T-2003-507739.